Monoclonal antibodies were once thought to be an ideal way to target malignant tissues, by delivering a killing agent, while leaving healthy tissue intact. However, their clinical potential is often limited due to the need to covalently couple the killing agent to the monoclonal antibody. Thus, in an effort to alleviate such limitations, bispecific antibodies were developed, which remain bivalent, but are specific for a target cell on one arm of the antibody and a killing agent on the other arm. The killing agent can be a toxin, a drug, a chelated radioisotope, or, more preferably, a cytotoxic effector cell.
Monoclonal antibodies can also show therapeutic activity against specific cells, e.g., malignant tissues based on the interaction of the Fc portion of the antibody heavy chain with other components of the immune system, such as the complement cascade or by binding to Fcγ receptors or various cytotoxic effector cell types.
Another means of effecting cell death comprises inducing the cross-linking of membrane antigens. Previous studies have indicated that antibody cross-linking of membrane B-cell markers (e.g., surface IgM, Valentine et al., Eur. J. Immunol. 22:3141 (1992); and MHC class II, Newell et al., PNAS 90:10459 (1993)) can inhibit malignant B cell proliferation and in many cases induce apoptosis (e.g., programmed cell death) in vitro.
Shan et al. (Blood 91:1644-1653) demonstrated that hyper-cross-linking of the CD20 antigen, by using the murine 1F5 antibody cross-linked with a goat anti-mouse IgG, inhibited growth of several human B-lymphoma cell lines in vitro. Similar results have now been published for both CD19 and CD22 when cross-linking of membrane bound MAb was amplified with a anti-mouse IgG (Chaouchi et al., J. Immunol. 154:3096 (1995)).
It may be possible that hyper cross-linking of these surface membrane markers could augment the existing anti-tumor activities of MAb's like C2B8, a chimeric monoclonal antibody specific for CD20, and increase therapeutic effectiveness. Therefore, molecules that can induce cell death in a pharmaceutically acceptable format would potentially provide an attractive therapeutic agent for immunotherapy of neoplastic disease.
Apparently with that goal in mind, Wolff et al. (Cancer Res. 53:2560-2565 (1993)) and Ghetie (PNAS 94:7509-7514 (1997)) have reported the chemical synthesis of several IgG/IgG homodimers to carcinoma associated surface antigen (BR96 and HER-2). The Ghetie dimers also included antibodies to several human B-cell markers (CD20, CD19, CD21, CD22). In this approach, one portion of the molecule was functionalized using a linker designed to introduce a reactive thiol on the antibody, while the other Ab portion used a linker to introduce a maleimido group. When purified from unreacted linkers and mixed together, the two antibodies complex by formation of a thioether (non-reducible) bridge that links the two IgG molecules, and forming a 300 kDa, tetravalent antibody (H4L4) molecule.
However, unfortunately, the yields of the 300 kDa IgG-homodimer were very low (20-25%) and were similar or lower than the CD19 homodimer, which ranged from 20-30% (Ghetie et al., PNAS 94:7509-7514 (1997)). Reducing SDS-PAGE gels of purified homodimer showed only a small percentage was linked via a thioether bond, indicating most of the dimers formed using this methodology may have been naturally occurring or mediated through disulfide bridging. Nevertheless, all of the purified dimers were growth inhibitory, although only the anti-carcinoma (Her-2) dimer and not homodimers directed against B cell markers CD19, CD20, CD21, CD22 were reported to be apoptotic. Additionally, the anti-CD19 homodimer was tested in animal models and shown to have anti-tumor activity. However, there is a need in the art for a more efficient method for producing homodimers, in particular for homodimers or heterodimers that are capable of initiating apoptosis, e.g., in proliferating malignant B-cells populations.
Among the problems associated with bispecific or dimeric antibodies is the response generated by the human immune system itself, which may respond to the antibody as a foreign agent. For instance, patients treated with drugs or radionuclides complexed with murine monoclonal antibodies (which have been the most commonly used targeting antibodies for human) develop circulating human anti-mouse antibodies (HAMAs) and a generalized immediate type-III hypersensitivity reaction to the antibody moiety of the conjugate. This problem is compounded when such antibodies are chemically linked. Furthermore, even when adverse side effects are minimal (for example, as in a single administration), circulating HAMAs decrease the effective concentration of the targeting agent in the patient and therefore limiting the bispecific antibody from reaching the target site.
As such, it is an object of the present invention to provide low toxicity compounds that may be used to target neoplastic cells.
It is another object of the invention to provide compounds that may effectively used to treat immune disorders.
It is still another object of the present invention to provide dimeric antibodies having high binding affinities and low immunogenicity.